Exposures to tobacco and other smoke, either direct or environmental, cause long-term harm through epigenetic effects on DNA methylation, but there are two major problems in the field. First, testing causality for exposure-related methylation changes identified epidemiologically has been impossible for methodologic reasons. Namely, there has been no way to specifically demethylate a putative epigenetic target sequence and then test predicted effects on gene expression and phenotype. Second, there is a need for novel therapeutic strategies to specifically reverse the epigenetic changes linked to environmental exposures. We propose to employ an innovative methodology we designed to epigenetically manipulate human club cell 16 (CC16) gene, a potentially beneficial gene dampened by methylation in chronic obstructive pulmonary disease (COPD), an outcome of many environmental injuries including smoke. For this, we aim to accomplish epigenetic re-activation of CC16 in human lung cell lines and primary cells using our novel method of targeted DNA demethylation. We have designed fusion complexes of demethylases thymine-DNA- glycosylase (TDG) and ten-eleven translocation proteins (Tet) with DNA-binding domains (DBD) made of zinc- finger protein arrays (ZFA), which provide the targeting precision needed to advance this approach. Specifically, we will optimize targeted demethylation of CC16 promoter in BEAS2B cell line (A549 as an alternative) via fusion protein constructs in which TDG or Tet's are fused with arrays of custom-built ZFAs targeting the CC16 promoter. Control constructs will include catalytically inactive enzymes (without demethylase activity) and ZFAs alone. The predictions are that the culture will show increased transcriptional responsiveness of CC16 and diminished DNA demethylation. The predicted specificity of the effect will be evaluated by expression array profiling. We will then test the phenotypic benefit from this upregulation after i vitro exposure of the cells to cigarette smoke extract by measuring several biomarkers of activation and apoptosis. In a subaim we propose a critical demonstration that could help advance this approach to translation: vector-free delivery of the fusion demethylases produced as proteins. Because transcriptional responsiveness to demethylation may vary in different cell types, and to increase translational potential of the study we will explore the effect of CC16 demethylation in human small airway epithelial cells and in primary cells from patients with COPD, using similar approaches. As these cells have limited number of divisions, we aim to perform most of the optimizations using cell lines first. Successful completion of these studies will provide a platform for development of epigenetic therapeutics and experimental agents of this novel class.